Over the past two decades the use of phytochemicals, nutraceuticals and other herbal products have increased damatically in part due to their low level of side effects while providing health benefits. Expanding research and anecdotal reports of their benefits have convinced the healthcare community and the general population that naturally-derived phytochemicals have the ability to combat diseases including diabetes, HIV, inflammation, cancer, obesity and toxicant-induced organ injuries.1
Among all diseases, the obesity epidemic may be the most serious, and has attracted much attention because it has been convincingly characterized as one of the root causes of other secondary diseases such as diabetes, hypertension and cardiovascular diseases. To remedy obesity alone, the healthcare field has spent an incredible amount of resources to look for novel conventional medications and surgical procedures although overall success has been very disappointing. An alternative approach has been to use phytochemicals or dietary supplements with anti-obese properties. This approach has shown great promise and is widely applied by the general public including healthcare professionals.
STG was used in this study keeping in focus its ability to promote body's energy expenditure. Of the two doses used, 1X and 7X, the higher dose showed a modest but significant weight loss in male rats at the end of 4 months ( and ), whereas female rats showed no response. Several possible explanations may exist for this observation. The differences in metabolism due to gender-specific hormone profiles, very short estrus cycle of rats (every 4 days), and length of dose exposure of STG which may not have been adequate for this species to show a desired effect at the human equivalent dose may have played a role. Although it is difficult to pin point a single reason for the differences in an in vivo model, the phytochemicals in STG are now commonly used by reputable laboratories to understand cellular energy-linked mechanisms and in clinical settings to translate experimental observations to weight loss protocols.
Investigators have shown that consumption of oolong tea in combination with EGCG containing guarana caused greater energy expenditure and fat oxidation in men.54
Komatsu et al.46
reported that women who consumed oolong tea after meal increased energy expenditure by 10% compared to an energy expenditure of 4% for green tea drinkers and 0% for water drinkers. Oolong tea consumption prior to eating carbohydrate-rich foods curb increases in insulin,31
thus reducing some of the fat-enhancing tendencies of carbohydrate intake, and consumption of the same product for weeks opposes obesity.31
It has also been suggested that tea and its components may influence glucose metabolism and diabetic hyperglycemia through several mechanisms, such as enhancing insulin sensitivity, and some human clinical studies have shown improvement in glucoregulatory control and endothelial function.55
Oolong tea leaf extracts also contain essential vitamins including A, B complex and C, and several minerals. STG's ingredients in the presence of vitamins and minerals may have additional boosting effects on the energy expenditure or the antioxidant system. Cumulatively, all these effects suggest that oolong tea components exert control over select metabolic pathways.55
Pin-pointing mechanisms of actions of these naturally occurring agents remains a major challenge because of one or more of the following reasons: (1) in vitro results often do not reproduce in vivo; (2) results derived from experimental animals may not accurately reproduce in humans; (3) a wide range of variations in human and experimental settings are often inconclusive; (iv) extrapolation of animal exposure data does not accurately suggest adequate daily intake levels in humans; and (v) bioavailability issues are additional confounding factors. The bioavailability of active components is beginning to be understood, but further research is required to determine whether the results from animal studies can be extrapolated to human setting. Nevertheless, a number of phytochemicals have progressed into clinical trials although major knowledge gaps remain to be filled. Transgenics and humanized experimental models are additional instruments that are now frequently used to understand intricacies that often do not surface and remain unnoticed during experimentation.
The second series of experiments examined specific serum markers to ascertain the safety of STG relative to vital target organs in the body ( and ). ALT and AST were used for the liver, BUN and creatinine were used for the kidneys and CK/LDH were used as biomarkers for the heart to determine any adverse influence of STG. The results indicated that STG produced slightly but not significantly elevated ALT, AST and CK activities over control values in female rats and similar increases only for CK in male rats, at 2 months of treatment.
Other biochemical markers including serum glucose, bilirubin, serum lipids and electrolytes as well as C-reactive protein and homocysteine ( and ) were found to be near control or below control values indicating no injury to the liver, kidneys and the heart or other tissues. Previous studies have demonstrated the absence of organ toxicity by extracts of guarana41
Sage has also been shown to have no effect on ALT and AST, indicators of hepatotoxicity, in humans.57
These effects of STG clearly establish safety and possibly bioavailability to three major organs and other tissues in the body.
Another series of experiments determined the influence of STG on tissue oxidative stress (–
) and how it modulated antioxidant pathways to minimize stress (–
) in all the organs. STG did not exacerbate the stress levels in any of these tissues, but rather quantifiably reduced oxidative stress in the liver and heart (–
) while kidneys were unaffected (). These results agree with previous studies demonstrating that sage extracts suppressed lipid peroxidation.16
Figure 4 Four months exposure of various doses of STG reduced cardiac oxidative stress (interpreted as % malondialdehyde accumulation resulting from lipid peroxidation). Oxidative stress in the heart indirectly reflects free radical production and its consequence, (more ...)
Radical-mediated lipid peroxidation is the key to membrane injury and subsequent MDA release. MDA concentrations determined in various tissues indirectly reflect the degree of free radical mediated lipid peroxidation which is a classic indicator for cytotoxic pathways. Minimization of this event is conducive to cellular survival and normal growth. Furthermore, the fact that organ specific serum chemistry markers () did not increase after STG exposure indirectly reflected either minimal or below normal production of free radicals in these tissues. Whether the observed effect was a direct interaction of ingredients of STG with the free radicals was not investigated. Besides lipid peroxidation, oxidative stress is considered the root cause of most macromolecular injury and minimization of such stress is beneficial to vital organs. Since most xenobiotics are naturally routed through the liver for biotransformation, occasionally it becomes an accidental victim. Overall, serum chemistry profiles corroborated the oxidative stress data, demonstrating little change in serum lipid and protein profiles in response to STG. Sage extract has been shown to decrease plasma LDL cholesterol and total cholesterol while increasing HDL in humans57
The next series of experiments assessed the effect of STG on various antioxidant elements that play a key role in the cellular defense (–
). The results indicated a differential effect in the various organs. STG enhanced SOD activity in all the three organs (–
), although liver showed the greatest increase in a dose response manner and kidneys exhibited the lowest response. Similarly, all the three organs showed an increase in glutathione peroxidase activity in response to STG (–
), with liver producing the highest and the kidneys the lowest increase. As far as the total glutathione is concerned, again liver was the best responder to STG and kidney was the least (–
). Male rats responded to STG better than the female rats. These parameters mirrored unanticipated but beneficial effects of STG on these prime components of the tissue defense. These results clearly mirrored the serum chemistry profiles and patterns of oxidative stress, and agree with previous reports where sage extracts increased glutathione levels.17
Numerous studies have shown that the consequences of uncontrolled production of free radicals due to malnutrition, stress, depletion of cellular antioxidants due to deregulated metabolism, and free radical mediated global oxidation of vital macromolecules are prime contributors to diseases. To circumvent these issues, investigators have devised ways to artificially maneuver cellular or organ-level regulation of glutathione, vitamin C, vitamin E, micronutrient selenium and some of the antioxidant enzymes, such as catalase, peroxidase and glutathione peroxidase. These efforts have resulted in a number of successful oxidativestress related disease fighting strategies coupled with many failures. Moreover, a universal strategy has not been found.
Over the last two decades, nutrition experts in the field have begun to recognize that naturally-derived antioxidants are significantly healthier and the myriad of phytochemicals commonly found in fruits, vegetables and edible plants are naturally designed with inherent antioxidant and chemoprotective properties. Furthermore, they rarely exhibit serious side effects and are excellent candidates for maintaining the intracellular and extracellular redox environment. This concept has impacted the basic approach to healthcare which is reflected in the continued growth in the worldwide sales of natural products in recent years.
Phytochemicals often alter cellular functions in a specialized manner without jeopardizing macromolecular conformations. Occasionally, the observed effect of a phytochemical may not be its direct effect but rather an indirect action of its metabolite(s), and many phytochemicals including those in STG have the ability to modulate biochemical events at the organ, cellular, subcellular and molecular levels.
STG was formulated such that all of its components work together and take advantage of some of these biochemical events related to free radicals and oxidative stress in various intracellular compartments. For example, polyphenols found in green tea enhance antioxidant (glutathione peroxidase, catalase and quinone reductase) and phase II (glutathione-S
-transferase) enzyme activities, inhibit chemically induced lipid peroxidation, inhibit irradiation-and TPA-induced epidermal ornithine decarboxylase (ODC) and cyclooxygenase activities.56
Similar effects have also been reported for sage and guarana extracts.24
Consistent with the above reports, in this study, STG was found to be an excellent inducer of all the key players of the antioxidant team.
Overall length of STG exposure or gender did not signifi- cantly influence the normal functioning of the target organs but did show major differences among some of the antioxidant components. While serum chemistry parameters and the oxidative stress are under the influence of free radicals, induction or inhibition of antioxidant elements are under strict genetic control. The fact that SOD, GPx and glutathione showed dramatic changes under the influence of STG suggests that the components of STG were bioavailable and accessible to intracellular compartments. However, it can not be speculated whether the components of STG acted singly or synergistically to exhibit these profound biochemical changes but experiments are in progress in our laboratories to determine such responses. The ability of the various components of STG to interact with the genome to exhibit such an effect can not be ruled out.
Numerous examples of genomic regulation by nutrients (nutrigenomics) have been published in recent years. Pycnogenol exerts its anticytotoxic property by stimulating glutathione biosynthesis.61
Rosemary extract was found to activate an array of detoxification enzymes including glutathione-S-transferase, and NAD(P)H: quinone reductase in the lung, liver and stomach.62
Cinnamon and coriander seed extracts increase superoxide dismutase, catalase, GST, glucose-6-phosphate dehydrogenase and glutathione-disulfide reductase activities in the liver.63
Fisetin, galangin, quercetin, kaempferol and genistein exhibit potent non-competitive inhibition of sulfotransferase 1A1.64
Various phytochemicals are inducers of CYP450-dependent drug metabolism, whereas some others are potent inhibitors.65
The impact on the CYP450 system is extremely important from a clinical perspective since co-exposure to these entities along with selected drugs can significantly influence the therapeutic outcome of a drug. Conversely, some drugs may adversely affect biochemical pathways normally under the influence of dietary phytochemicals, resulting in unwanted effects. Examples include the ability of several phytochemicals to interfere with the cell cycle regulatory elements (carcinogen bio-activation, angiogenesis and inflammation) and cancer signaling pathways.72
All these reports strongly suggest abilities of phytochemicals to interact with the genome and influence the cell globally.
Since the late 1990s, our laboratory has conducted mechanistic experiments investigating the anti-toxic, anti-cancer, anti-apoptotic and anti-necrotic properties of grape seed proanthocyanidin extract, quercetin, rutin, hesperitin and Momordica charantia
and Ocimum sanctum
extracts in in vivo models and has elucidated unique organoprotective pathways when these phytochemicals act alone or in combination.5
Protection of the genomic integrity is one of the most aggressively pursued goal of a cell. Flawed genomic machinery invariably leads to deregulated metabolism. In order to verify the integrity of the genome in all the three vital organs under the influence of STG, total cellular DNA fragmentation assays were performed (–
). Results indicate that STG did not cause genomic injury or any type of genomic instability. The lack of DNA damage (fragmentation) does not tell us whether the large increases in SOD and GPx activities observed in this study in response to STG are due to a genetically-driven induction of new enzymes or an increase in enzyme activities. Nevertheless, this outcome is generally considered beneficial for the organism. Furthermore, both oolong tea extract28
and sage extract17
have been shown to inhibit DNA damage, supporting the results of this study with STG. Oxidative-stress mediated genomic injury and its prevention in phytochemical pre-exposed liver, kidneys and heart has already been reported.7
In addition to all these parameters, histopathological diagnosis of H&E and PAS-stained organ sections clearly mirrored the serum chemistry changes, and strongly correlated with some of the biochemical parameters. Gross tissue morphology indirectly reflected untainted metabolic status in all the three organs. Representative sections exposed to the 7x dose of STG are shown (–; and ). Organ sections from control or STG-exposed animals resembled each other with unperturbed tissue architecture. Multiple sections were thoroughly examined to rule the artifactual changes that may have resulted due to tissue processing. Uniform stain intensity reflected health of the cells and indirectly discerned intactness of the intracellular organelles. Nuclear, cytoplasmic and outer boundaries of the hepatocytes were intact. A close examination of kidney sections disclosed normal features of proximal tubular cells, distal tubular cells and glomerular apparatus. Examination of heart sections showed normal skeletal muscle fibers, normal intercalated discs and connective tissue, normal nuclei and scattered fibrocytes. None of the areas in any of the target organs revealed the presence of any inflammatory cells, indicating the absence of any inflammation. Apoptotic cells were rarely found in normal or STG-exposed tissues. All these features clearly established the safety of STG. The general health of the animals was another indicator which was closely monitored during the entire study. Control and STG-exposed animals did not show any signs of illness.
In conclusion, 4-month exposure of male and female rats to STG dramatically enhanced antioxidant power in the absence of any genomic injury to the liver, heart and kidneys. Significant changes in antioxidant components indirectly disclosed absorption, biodistribution and bioavailability of STG at least in these three organs and liver showed the best response. Although STG exposure did not drastically reduce weight gain, it did help maintain healthy body weight coupled with a robust antioxidant capacity of the vital target organs.